Alkaline phosphatase

From Wikipedia, the free encyclopedia
Jump to: navigation, search
Alkaline phosphatase
1ALK.png
Ribbon diagram (rainbow-color, N-terminus = blue, C-terminus = red) of the dimeric structure of bacterial alkaline phosphatase.[1]
Identifiers
EC number 3.1.3.1
CAS number 9001-78-9
Databases
IntEnz IntEnz view
BRENDA BRENDA entry
ExPASy NiceZyme view
KEGG KEGG entry
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Gene Ontology AmiGO / EGO
Alkaline phosphatase
PDB 1alk EBI.jpg
Structure of alkaline phosphatase.[1]
Identifiers
Symbol Alk_phosphatase
Pfam PF00245
InterPro IPR001952
SMART SM00098
PROSITE PDOC00113
SCOP 1alk
SUPERFAMILY 1alk

Alkaline phosphatase (ALP, ALKP) (EC 3.1.3.1) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation. As the name suggests, alkaline phosphatases are most effective in an alkaline environment. It is sometimes used synonymously as basic phosphatase.[2]

Bacterial[edit]

In Gram-negative bacteria, alkaline phosphatase is located in the periplasmic space, external to the cell membrane. Since this space is much more subject to environmental variation than the actual interior of the cell, bacterial alkaline phosphatase is comparatively resistant to inactivation, denaturation, and degradation, and also has a higher rate of activity. Although the purpose of the enzyme is not fully resolved, the simple hypothesis that it is a means for the bacteria to generate free phosphate groups for uptake and use is supported by the fact that alkaline phosphatase is usually produced by the bacteria only during phosphate starvation and not when phosphate is plentiful.[3] However, other possibilities exist. For instance, the presence of phosphate groups usually prevents organic molecules from passing through the membrane; therefore, dephosphorylating them may be important for bacterial uptake of organic compounds in the wild.[4] Some complexities of bacterial regulation and metabolism suggest that other, more subtle, purposes for the enzyme may also play a role for the cell. In the laboratory, however, mutant Escherichia coli lacking alkaline phosphatase survive quite well, as do mutants unable to shut off alkaline phosphatase production.[5]

The optimal pH for the activity of the E. coli enzyme is 8.0[6] while the bovine enzyme optimum pH is slightly higher at 8.5.[7]

Use in research[edit]

Typical use in the lab for alkaline phosphatases includes removing phosphate monoester to prevent self-ligation.[8]

Common alkaline phosphatases used in research include:

  • Shrimp alkaline phosphatase (SAP), from a species of Arctic shrimp (Pandalus borealis). This phosphatase is easily inactivated by heat, a useful feature in some applications.
  • Calf-intestinal alkaline phosphatase (CIP)
  • Placental alkaline phosphatase (PALP) and its C terminally truncated version that lacks the last 24 amino acids (constituting the domain that targets for GPI membrane anchoring) - the secreted alkaline phosphatase (SEAP)

Alkaline phosphatase has become a useful tool in molecular biology laboratories, since DNA normally possesses phosphate groups on the 5' end. Removing these phosphates prevents the DNA from ligating (the 5' end attaching to the 3' end), thereby keeping DNA molecules linear until the next step of the process for which they are being prepared; also, removal of the phosphate groups allows radiolabeling (replacement by radioactive phosphate groups) in order to measure the presence of the labeled DNA through further steps in the process or experiment. For these purposes, the alkaline phosphatase from shrimp is the most useful, as it is the easiest to inactivate once it has done its job.

Another important use of alkaline phosphatase is as a label for enzyme immunoassays.

Undifferentiated pluripotent stem cells have elevated levels of alkaline phosphatase on their cell membrane, therefore alkaline phosphatase staining is used to detect these cells and to test pluripotency (i.e., embryonic stem cells or embryonal carcinoma cells).[9]

Dairy Industry[edit]

Alkaline phostphatase is commonly used in the dairy industry as an indicator of successful pasteurization. This is because the most heat stable bacterium found in milk, Mycobacterium paratuberculosis, is destroyed by temperatures lower than those required to denature ALP. Therefore ALP presence is ideal for indicating successful pasteurization.[10][11]

Pasteurization verification is typically performed by measuring the fluorescence of a solution which becomes fluorescent when exposed to active ALP. Fluorimetry assays are required by milk producers in the UK to prove alkaline phosphatase has been denatured,[12] as p-Nitrophenylphosphate tests are not considered accurate enough to meet health standards.

Alternatively the colour change of a para-Nitrophenylphosphate substrate in a buffered solution (Aschaffenburg Mullen Test) can be used.[13] Raw milk would typically produce a yellow colouration within a couple of minutes, whereas properly pasteurised milk should show no change. There are exceptions to this, as in the case of heat-stable alkaline phophatases produced by some bacteria but these bacteria should not be present in milk.

Inhibitors[edit]

All mammalian alkaline phosphatase isoenzymes except placental (PALP and SEAP) are inhibited by homoarginine, and, in similar manner, all except the intestinal and placental ones are blocked by levamisole. Heating for ~2 hours at 65 °C inactivated most isoenzymes except Placental isoforms (PALP and SEAP).[14]

Human[edit]

Physiology[edit]

In humans, alkaline phosphatase is present in all tissues throughout the entire body, but is particularly concentrated in liver, bile duct, kidney, bone, and the placenta. Humans and most other mammals contain the following alkaline phosphatase isozymes:

  • ALPI – intestinal (Molecular weight of 150 MDa)
  • ALPL – tissue-nonspecific (liver/bone/kidney)
  • ALPP – placental (Regan isozyme)

Diagnostic use[edit]

Normal ALP levels in adults are approximately 20 to 140 IU/L,[15] though levels are significantly higher in children and pregnant women. Blood tests should always be interpreted using the reference range from the laboratory that performed the test. High ALP levels can occur if the bile ducts are obstructed.[16] Also, ALP increases if there is active bone formation occurring, as ALP is a byproduct of osteoblast activity (such as the case in Paget's disease of bone). Levels are also elevated in people with untreated Coeliac disease.[17] Lowered levels of ALP are less common than elevated levels. The source of elevated ALP levels can be deduced by obtaining serum levels of gamma glutamyltransferase (GGT). Concomitant increases of ALP with GGT should raise the suspicion of hepatobiliary disease.[18]

Elevated levels[edit]

If it is unclear why alkaline phosphatase is elevated, isoenzyme studies using electrophoresis can confirm the source of the ALP. Heat stability also distinguishes bone and liver isoenzymes ("bone burns, liver lasts"). Placental alkaline phosphatase is elevated in seminomas[19] and active form of rickets, as well as in the following diseases:[20]

Lowered levels[edit]

The following conditions or diseases may lead to reduced levels of alkaline phosphatase:

In addition, the following drugs have been demonstrated to reduce alkaline phosphatase:

  • Oral contraceptives[21]

Leukocyte alkaline phosphatase[edit]

Leukocyte alkaline phosphatase (LAP) is found within white blood cells. White blood cell levels of LAP can help in the diagnosis of certain conditions.

See also[edit]

References[edit]

  1. ^ a b PDB 1ALK: Kim EE, Wyckoff HW (March 1991). "Reaction mechanism of alkaline phosphatase based on crystal structures. Two-metal ion catalysis". J. Mol. Biol. 218 (2): 449–64. doi:10.1016/0022-2836(91)90724-K. PMID 2010919. 
  2. ^ Tamás L, Huttová J, Mistrk I, Kogan G (2002). "Effect of Carboxymethyl Chitin-Glucan on the Activity of Some Hydrolytic Enzymes in Maize Plants". Chem. Pap. 56 (5): 326–329. 
  3. ^ Horiuchi T, Horiuchi S, Mizuno D (May 1959). "A possible negative feedback phenomenon controlling formation of alkaline phosphomonoesterase in Escherichia coli". Nature 183 (4674): 1529–30. doi:10.1038/1831529b0. PMID 13666805. 
  4. ^ Ammerman JW, Azam F (March 1985). "Bacterial 5-nucleotidase in aquatic ecosystems: a novel mechanism of phosphorus regeneration". Science 227 (4692): 1338–40. doi:10.1126/science.227.4692.1338. PMID 17793769. 
  5. ^ Wanner BL, Latterell P (October 1980). "Mutants affected in alkaline phosphatase, expression: evidence for multiple positive regulators of the phosphate regulon in Escherichia coli". Genetics 96 (2): 353–66. PMC 1214304. PMID 7021308. 
  6. ^ Garen A, Levinthal C (March 1960). "A fine-structure genetic and chemical study of the enzyme alkaline phosphatase of E. coli. I. Purification and characterization of alkaline phosphatase". Biochim. Biophys. Acta 38: 470–83. doi:10.1016/0006-3002(60)91282-8. PMID 13826559. 
  7. ^ Harada M, Udagawa N, Fukasawa K, Hiraoka BY, Mogi M (February 1986). "Inorganic pyrophosphatase activity of purified bovine pulp alkaline phosphatase at physiological pH". J. Dent. Res. 65 (2): 125–7. doi:10.1177/00220345860650020601. PMID 3003174. 
  8. ^ Maxam AM, Gilbert W (1980). "Sequencing end-labeled DNA with base-specific chemical cleavages". Meth. Enzymol. Methods in Enzymology 65 (1): 499–560. doi:10.1016/S0076-6879(80)65059-9. ISBN 978-0-12-181965-1. PMID 6246368. 
  9. ^ "Appendix E: Stem Cell Markers". Stem Cell Information. National Institutes of Health, U.S. Department of Health and Human Services. Retrieved 2013-09-24. 
  10. ^ Kay, H. (1935). "Some Results of the Application of a Simple Test for Efficiency of Pasteurisation". The Lancet 225 (5835): 1516–1518. doi:10.1016/S0140-6736(01)12532-8.  edit
  11. ^ Hoy, W. A.; Neave, F. K. (1937). "The Phosphatase Test for Efficient Pasteurisation". The Lancet 230 (5949): 595. doi:10.1016/S0140-6736(00)83378-4.  edit
  12. ^ BS EN ISO 11816-1:2013
  13. ^ Aschaffenburg R, Mullen JEC (1949). "A rapid and simple phosphatase test for milk". Journal of Dairy Research 16 (1): 58–67. doi:10.1017/S0022029900005288. 
  14. ^ Alkaline Phosphatase Why It Is Done from Everday Health.com. Retrieved October 15, 2012.
  15. ^ "MedlinePlus Medical Encyclopedia: ALP isoenzyme test". 
  16. ^ ALP: The Test
  17. ^ Preussner, Harold T, HT (March 1998). "Detecting coeliac disease in your patients". American Family Physician 57 (5): 1023–1034. PMID 9518950. 
  18. ^ Vroon, David. "Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd edition". 
  19. ^ Lange PH, Millan JL, Stigbrand T, Vessella RL, Ruoslahti E, Fishman WH (August 1982). "Placental alkaline phosphatase as a tumor marker for seminoma". Cancer Res. 42 (8): 3244–7. PMID 7093962. 
  20. ^ Dugdale, David C. "ALP-bloodtest:MedlinePlus Medical Encyclopedia". MedlinePlus. Retrieved 2014-02-26. 
  21. ^ Schiele F, Vincent-Viry M, Fournier B, Starck M, Siest G (November 1998). "Biological effects of eleven combined oral contraceptives on serum triglycerides, gamma-glutamyltransferase, alkaline phosphatase, bilirubin and other biochemical variables". Clin. Chem. Lab. Med. 36 (11): 871–8. doi:10.1515/CCLM.1998.153. PMID 9877094. 
  22. ^ Arceci RJ, Hann IM, Smith OP, ed. (2006). Pediatric hematology (3rd ed.). Wiley-Blackwell. p. 763. ISBN 978-1-4051-3400-2. 

External links[edit]

Further reading[edit]